ES2698570T3 - Methods and compositions related to p62 for the treatment and prophylaxis of cancer - Google Patents

Abstract

An agent that comprises: a. at least 30 consecutive amino acids of a p62 / SQSTM1 polypeptide; b. a nucleic acid encoding p62 / SQSTM1, wherein said nucleic acid encoding p62 / SQSTM1 encodes at least 30 consecutive amino acids of a p62 / SQSTM1 polypeptide; c. a p62 / SQSTM1 polypeptide at least 90% identical to SEQ ID NO. two; d. a p62 / SQSTM1 polypeptide with at least one or more domain deletions; and. a p62 / SQSTM1 nucleic acid encoding a polypeptide at least 90% identical to SEQ ID NO: 2; or f. a p62 / SQSTM1 nucleic acid encoding a polypeptide with at least one or more domain deletions; for use in the treatment, relief, improvement, palliation, delay in onset, inhibition of progression, reduction of severity and / or reduction of the incidence of one or more symptoms of a cancer in a subject.

Description

DESCRIPTION

Methods and compositions related to p62 for the treatment and prophylaxis of cancer

Cross-reference to related patent applications

Field of the invention

This invention relates in general to the field of cancer prevention and treatment. More specifically, the invention relates to the prevention and treatment of cancer by activating the cancer response using compositions of p62.

BACKGROUND OF THE INVENTION

Cancer is the second most common cause of death in the United States and the European Union (National Vital Statistics Reports, vol 60, No. 4, 2012) and the first most common cause of death among people aged 45 to 64 years, for both men and women, in the European Union. The estimated prevalence of cancer in the United States as of January 1, 2008 was 5,506,000 cases of invasive tumors for men and 6,452,000 cases for women.

Cancer vaccines are under investigation, with some in Phase III efficacy studies (Rosenberg et al., Nat Med., 10: 909 (2004), Johnson et al., Expert Rev Anticancer Ther., 9:67 ( 2009) 3,4). Several cancer vaccines are directed against solid tumors: melanoma, prostate, lung, breast and colorectal cancer. Two high-risk populations will particularly benefit from preventive vaccines against cancer: patients with surgically excised tumors that belong to types of cancer that are known to have a high metastatic potential (eg, ovarian or some types of breast cancer) and also carriers of known mutations, associated with an increased risk of cancer (eg, mutations in the BRCA1 and BRCA2 genes for breast and ovarian cancers, RAD51 gene for ovarian cancer, etc.). Selective preventive vaccination of high-risk women is an important public health task.

p62 is a multifunctional protein that binds to ubiquitin and regulates the activation of the signaling pathway of nuclear factor kappa-B (NF-kB). The protein functions as a structure / adapter protein in concert with factor 6 associated with the TNF receptor to mediate the activation of NF-kB in response to signals upstream. Alternatively, spliced transcript variants encoding isoforms identical or different for this gene have been identified.

p62 was identified as a 62 kDa protein that bound to the src 2 homology domain (SH2) of Lckp56 tyrosine kinase independently of phosphotyrosine (Park et al., Proc Natl Acad Sci USA., 92: 12338 (1995 )). The primary sequence of p62 is known (Joung et al., Proc Natl Acad Sci USA., 93: 5991, (1996)), and it was shown to bind to ubiquitin (Vadlamudi et al., J. Biol. Chem. , 271: 20235 (1996)). The DNA sequence of human p62, sequence of Homo sapiens sequestosome 1 (SQSTM1), transcript variant 1, mRNA, can be accessed in the NCBI reference sequence: ⁿ M_003900.4. The amino acid sequence of human p62, isquorm 1 of the sequestosome-1 [Homo sapiens], can be accessed in the NCBI reference sequence: NP_003891.1. p62 has no homology to C-terminal hydrolases of ubiquitin or to the S5a subunit of the 26 S proteasome complex, the only known proteins that bind to ubiquitin non-covalently. These results suggest that p62 belongs to a new class of ubiquitin binding proteins.

P62 is a component of the inclusion bodies that is found in protein aggregation diseases in the brain and liver: p62 is sequestered in cytoplasmic inclusion bodies, called sequestosomes. These aggregates of p62-containing proteins are degraded by autophagy. It was suggested that this function of p62 may have a protective effect on cell death induced by huntingtin (Bj ^ rk ^ et al., J Cell Biol., 171: 603 (2005)). Mutations in the p62 gene have been associated with familial and sporadic Paget's disease (Jenny Chung et al., Semin Arthritis Rheum., 41: 619 (2012)), a metabolic bone disease.

Summary of the invention

In the present document methods are provided for treating, alleviating, improving, alleviating, delaying the onset, inhibiting progression, reducing the severity and / or reducing the incidence of one or more symptoms of a cancer in a subject by administering to the subject an agent that has (a) a p62 polypeptide or (b) a nucleic acid encoding p62. The agent may be (a) one or more deletions domain, (b) a nucleic acid encoding p62 is at least 95% identical to ^s E ^q ID NO. 1, or (c) a p62 polypeptide that is at least 98% identical to S ^e Q ID NO: 2. Removals from the domain of the method can be one or more of the following: PB1, ZZ, NLS2, TB, NLS1, NES, LIR, KIR and UBA. The method can use a fusion polypeptide or a nucleic acid encoding a fusion polypeptide, respectively. The method can use a p62 polypeptide that is modified after translation.

The agent can be administered by any of the following routes: parenterally, orally, nasally, rectally, transdermally, intravaginally or inhaled through an aerosol. Parenteral routes can be any of the following: intravascular, intravenous, intraarterial, intramuscular, intraocular, intraperitoneal, intradermal and subcutaneous, or can be administered to an organ or tumor. The method may further include any and all of the following: administration of adjuvants, administration of costimulatory components, administration of one or more molecules that block suppressive or negative regulatory immune mechanisms, or administration of one or more anti-cancer therapies to said subject.

The method can be used to treat any type of cancer in a subject that includes: breast cancer, lung cancer, prostate cancer, gastric cancer, colorectal cancer, skin cancer, head and neck cancer, bronchial cancer, cancer pancreas, urinary bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, cancer of the biliary tract , cancer of the small intestine or appendix, ovarian cancer, cancer of the uterus, cancer of the salivary gland, cancer of the thyroid gland, cancer of the thyroid gland, cancer of the adrenal gland, osteosarcoma, chondrosarcoma, sarcoma and cancer of the hematological tissues. The subject may be: a subject diagnosed with cancer, a subject previously treated for cancer, a subject with a family history of cancer or a subject predisposed to cancer.

The method can include an agent which is a nucleic acid encoding p62 and the nucleic acid can be included in a plasmid or a viral vector. The method may also include a strategy for improving the efficiency of nucleic acid-based immunization.

Also provided in this document are agents for treating, alleviating, ameliorating, calming, delaying the onset, inhibiting progression, reducing the severity and / or reducing the incidence of one or more symptoms of a cancer in a subject that is a p62 or a nucleic acid encoding p62, having at least one or more domain deletions, or composed of one or more domains of a p62 polypeptide or a nucleic acid encoding one or more domains of a p62. The one or more domains can be between the following: PB1, ZZ, NLS2, TB, NLS1, NES, LIR, KIR and UBA. The agent may include a fusion polypeptide or a coding nucleic acid, respectively. The p62 polypeptide can be modified after translation.

The agent may further include one or more adjuvants, one or more costimulatory components, or one or more molecules that block suppressive or negative regulatory immune mechanisms, one or more chemotherapeutic molecules or anti-angiogenic molecules.

The agent can be a nucleic acid encoding p62 in addition which is a component of a plasmid or a viral vector.

Also included in this document are compositions that include the agent suitable for administration to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a natural p62 human nucleic acid sequence (SEQ ID NO: 1).

Fig. 2 shows a natural amino acid sequence of human p62 encoded by the nucleic acid sequence (SEQ ID NO: 2).

Fig. 3 shows a representation of the domain structure of human p62.

Fig. 4 shows a comparison of a time course of tumor formation in a breast cancer model in mice for mice injected with a p62 DNA vaccine, HER2 (positive control) or vector alone (negative control).

Fig. 5 shows hematoxylin and eosin (HE) staining of tumors from animals immunized with p62. The upper panel: the arrows point to multiple foci of necrosis. The lower panel: the arrows point to a mesh of inflammatory cells.

Fig. 6 shows the immunohistochemical staining of tumors from animals immunized with HER2 and p62. The panels on the left show HE staining, the central panels show anti-CD3 staining and the panels on the right show anti-CD11b staining.

Fig. 7 shows a graphical timeline of the administration of the p62 DNA vaccine in a rat T5 breast cancer model:

FIG. 8 shows a temporal course of tumor volume in rats vaccinated with p62 and control with T5 transplantable breast carcinoma.

Fig. 9 shows the time course of the inhibition of tumor growth in rats vaccinated with p62 and control with transplantable breast cancer with T5.

Fig. 10 shows a time course of rat survival in rats vaccinated with p62 and control with T5 transplantable breast carcinoma.

Fig. 11 shows sections of tumors of rats vaccinated with p62 and control vector with T5 transplantable breast carcinoma.

Fig. 12 shows hematoxylin and eosin (HE) staining of the tumor sections of rats vaccinated with p62 and control vector with T5 transplantable breast carcinoma.

Fig. 13 shows a comparison of the number of metastases of Lewis lung carcinoma among mice vaccinated with p62 and control vector.

Detailed description of the invention

In this document p62 compositions and methods for the treatment of cancer are provided. Although not wishing to be bound by any theory, the inventors have found that by administering the nucleic acid encoding p62 to a subject, the immune defense mechanism of the host is stimulated to attack the neoplastic cells. Accordingly, DNA vaccines encoding a p62 polypeptide, or p62 polypeptides, administered to a subject can stimulate an immune response against cancer. The present invention is defined by claims 1-15.

As used herein, "p62 polypeptide" means a polypeptide corresponding to the full length p62 / SQSTM1 protein. The term includes all homologs, analogs, fragments or derivatives of the p62 / SQSTM1 protein. In one embodiment, the isolated p62 polypeptide has an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2). A "p62-encoding nucleic acid" means a DNA or RNA that encodes a p62 polypeptide.

In some embodiments, the subject is a human being. In other embodiments, the subject is a non-human mammal, for example, a horse, a cow, a sheep, a pig, a deer, a dog, a cat, a rat or a mouse.

In addition to the full-length amino acid sequence, the polypeptides of the present invention may also include fragments or truncations, analogs and homologs of the p62 polypeptide and its truncations, as described herein. The fragments may include peptides of at least 5, at least 10, at least 15, at least 20, at least 25 or at least 30 amino acid residues of the full-length polypeptide.

Deletions of one or more amino acids, or discrete portions of the amino acid sequence of the p62 / SQSTM1 protein are also included. The eliminated amino acids may or may not be contiguous. The lower limit length of the resulting analogue with a deletion mutation is about 10, about 20, about 50 or about 100 amino acids.

In some embodiments, the p62 polypeptide has one or more deleted domains. While not wishing to be bound by any theory, the inventors contend that the removal of one or more domains of the p62 polypeptide provides a more compact and manipulable polypeptide to direct an immune response. For example, by interrupting or eliminating one or more of the domains of a p62 polypeptide, immunogenicity can be maintained (or improved, if the deleted or disrupted domain does not contribute to immunogenicity) in a more compact molecule and potentially increase the number of epitopes. presented to house antibodies on a basis by weight. In addition, the elimination or rearrangement of the domains responsible for the commitment to other cellular processes or their own degradation of intracellular proteins can improve their anticarcinogenic effect. The p62 polypeptide has a domain structure as provided in Table 1 below and as shown in FIG. 3:

Table 1. Structure of the p62 polypeptide domain

Sequence numbering: NP_003891 (isotherm 1 of sequestosome-1 [Homo sapiens]).

In some embodiments, one or more of the above domains are deleted from a p62 polypeptide at the corresponding codons for the nucleic acid regions of the p62 nucleic acid (deletions in frame), as presented below in Table 2.

Table 2. Eliminations in p62

The nucleotide numbers refer to the reference sequence of NCBI p62 NP_003891 (sequestosoma-1 isotherm 1 [Homo sapiens)].

For example, any deletion of the coding nucleic acid sequence starting at nucleotide 102 to nucleotide 122 and ending at 167 to 183 is considered a ZZ deletion. Therefore, for example, a 110 175 is a Z ^z elimination. Those skilled in the art are well aware of the techniques for creating in-frame eliminations. In some embodiments, the p62 polypeptide (or the p62 polypeptide encoded by a nucleic acid) is composed of one or more of the above domains. In some embodiments, the p62 polypeptide (or the p62 polypeptide encoded by a nucleic acid) is composed of two or more of the above domains and even more embodiments, the domains are those that make up the polypeptide in an N-to-C end order -terminal different than that presented in the natural p62 polypeptide.

As used herein, "biologically active or immunologically active" refers to polypeptides that have a similar structural function (but not necessarily to the same degree), and / or a similar regulatory function (but not necessarily to the same degree) , and / or similar biochemical function (but not necessarily to the same degree) and / or immunological activity (but not necessarily to the same degree) as the individual natural polypeptides.

As used herein, a "deletion" is defined as a change in the amino acid sequence in which one or more amino acid residues are absent as compared to the natural protein.

As used herein, an "insertion" or "addition" is a change in an amino acid sequence that has resulted in the addition of one or more amino acid residues as compared to the native protein.

As used herein, "substitution" results from the replacement of one or more amino acids by different amino acids, respectively, as compared to the natural protein. In some embodiments, the substitution mutation is C145R or Q418R.

As used herein, the term "variant" means any polypeptide having a substitution, deletion or addition of one (or more) amino acids of the sequence or sequence (or any combination thereof), including allelic variations, in comparison with the natural protein, provided that the protein result retains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the immunogenic activity compared to natural proteins as use in the present invention. Typically, variants of the polypeptides encompassed by the present invention will have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least less 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with respect to the SEQ. ID. DO NOT. two.

The sequence identity or homology can be determined using standard techniques known in the art, such as the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12; 387-395 (1984) or the BLASTX program (Altschul et al., J Mol. Biol. 215, 403-410). The alignment may include the insertion of gaps in the sequences to be aligned. In addition, for sequences that contain more or fewer amino acids than the proteins described herein, it is understood that the percentage of homology will be determined as a function of the number of homologous amino acids in relation to the total number of amino acids.

In some embodiments, the variants or derivatives of the polypeptides of the present invention maintain the hydrophobicity / hydrophilicity of the amino acid sequence. Conservative amino acid substitutions may be made, for example 1, 2 or 3 to 10, 20 or 30 substitutions, provided that the modified sequence retains the ability to act as an immunogen. Amino acid substitutions may include the use of non-natural analogues; for example, to increase the half-life in the blood plasma. Conservative substitutions are known in the art.

The term "derivative", as used herein in relation to the amino acid sequence, means the chemical modification of a polypeptide of the invention.

Non-limiting examples of such modifications may include, among others, aliphatic esters or amides of the carboxyl terminus or residues containing carboxyl side chains, O-acyl derivatives of residues containing hydroxyl groups and N-acyl derivatives of the residues they contain. amino-terminal amino acid groups or amino; for example, lysine or arginine.

Additional modifications may include, for example, the production of a polypeptide conjugated with polyethylene glycol (PEG), or the addition of PEG during the chemical synthesis of a polypeptide as described herein.

Modifications of the polypeptides or their portions may also include reduction / alkylation; chemical coupling to a suitable vehicle or treatment with mild formalin.

The term "modified", as used herein, refers to the presence of a post-translational modification in a polypeptide. The term "(modified)" means that the polypeptides being discussed are optionally modified; that is, the polypeptides under discussion can be modified or not modified. The term "modified after translation" and "modified" refers to any modification of a natural or non-natural amino acid that occurs in said amino acid after it has been incorporated into a polypeptide chain. The term encompasses, by way of example only, co-translational in vivo modifications, post-translational in vivo modifications, and post-translational in vitro modifications.

Other derivatives of the polypeptides of the present invention include incorporation of non-natural amino acid residues, or phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine or phosphothreonine residues. Other potential modifications include sulfonation, biotinylation or the addition of other moieties, particularly those that have molecular forms similar to phosphate groups.

The derivatives also include polypeptides modified by glycosylation. These can be done by modifying the glycosylation patterns during synthesis and processing in various alternative eukaryotic host expression systems, or during other processing steps. Methods for producing glycosylation modifications include exposing the p62 polypeptide to glycosylated enzymes derived from cells that normally carry out such processing, such as mammalian glycosylation enzymes. Alternatively, deglycosylation enzymes can be used to remove bound carbohydrates during production in eukaryotic expression systems. In addition, the coding sequence can also be modified so that the glycosylation sites are added or the glycosylation sites are deleted or disabled. In addition, if no glycosylation is desired, the proteins can be produced in a prokaryotic host expression system.

Variants and / or derivatives of the polypeptides of the invention can be prepared by chemical synthesis or by the use of site-directed mutagenesis (Gillman et al., Gene 8:81 (1979); Roberts et al, Nature 328: 731 (1987)). or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY) or the polymerase chain reaction method (PCR; Saiki et al, Science 239: 487 ( 1988)), as illustrated by Daugherty et al (Nucleic Acids Res. 19: 2471 (1991)) to modify the nucleic acids encoding the p62 polypeptides of the invention.

The polypeptides of the present invention may contain a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), the expression and / or secretion of the fusion protein can be increased by the use of a heterologous signal sequence. The signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion at one or more cleavage events. Such signal peptides contain processing sites that allow excision of the signal sequence of mature proteins as they pass through the secretory pathway. Therefore, the invention relates to the described polypeptides having a signal sequence, as well as to polypeptides from which the signal sequence has been proteolytically cleaved (ie, the cleavage products).

In order to improve the stability and / or reactivity, the polypeptides of the present invention can also be modified to incorporate one or more polymorphisms in the amino acid sequence resulting from the natural allelic variation. In addition, the D-amino acids, non-natural amino acids or non-amino acid analogs can be substituted or added to produce a modified p62 polypeptide.

The polypeptides of the present invention can be produced by the expression of a nucleotide sequence encoding them in a suitable expression system.

In addition, or alternatively, the polypeptides can be produced using chemical methods to synthesize the desired amino acid sequence, in whole or in part. For example, the polypeptides can be synthesized by solid phase techniques, cleaved from the resin and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures and Molecular Principles, WH Freeman and Co, New York N.Y.). The composition of the synthetic polypeptides can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). In addition, the amino acid sequence of a p62 polypeptide, or any of its parts, can be altered during direct synthesis and / or combined using chemical methods with a sequence of other subunits, or any of its parts, to produce a variant polypeptide.

Assays for measuring the immunological activity of any homologue, derivative or variant of any polypeptide of the present invention are well known in the art.

As used herein, the term "fusion proteins" refers to chimeric proteins that comprise amino acid sequences of two or more different proteins. Typically, the fusion proteins result from in vitro recombinant techniques well known in the art.

The fusion proteins of the present invention may further comprise one or more additional polypeptide domains added to facilitate the purification of proteins, to increase the expression of the recombinant protein, or to increase the solubility of the recombinant protein. Said domains that facilitate purification / expression / solubility include, among others, metal chelating peptides, such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3 .26328 1), domains of protein A that allow the purification of immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (Immunex Corp, Seattle, Washington). The inclusion of a cleavable linker sequence, such as Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and a p62 polypeptide is useful to facilitate purification.

Additional fusion expression vectors include pGEX (Pharmacia, Piscataway, NJ), pMAL (New England Biolabs, Beverly, Mass.) And pRITS (Pharmacia, Piscataway, NJ) that fuse glutathione S transferase (GST), the protein of binding to maltose B, or protein A, respectively, to the target recombinant protein. EBV, BKV and other episomal expression vectors (Invitrogen) can also be used.

In certain embodiments, a nucleic acid molecule encoding the p62 polypeptide is used. The nucleic acid molecule may comprise or consist of a nucleotide sequence encoding one or more p62 polypeptides, or fragments (including fragments encoding domains in any order or polypeptides in which one or more domains are deleted or interrupted) or their derivatives, such as those contained in a DNA insert in an ATCC deposit. The term "nucleic acid sequence" or "nucleic acid molecule" refers to a DNA or RNA sequence. The term encompasses molecules formed from any of the known DNA and RNA base analogs, such as, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil , 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methyl-guanine, 1-methyllinosine , 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D - mannosylkeosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudoouracil, kerosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, acid methyl ester N-uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, pseudouracil, kerosine, 2-thiocytosine and 2,6-diaminopurine, among others.

The vectors can be used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is used. An expression vector is a nucleic acid molecule that is suitable for introduction and / or propagation in a host cell and contains nucleic acid sequences that direct and / or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide. The flanking sequences may be homologous (i.e., of the same species and / or strain as the host cell), heterologous (i.e., of a species other than the species or strain of the host cell), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, for example.

A flanking sequence is preferably capable of effecting the replication, transcription and / or translation of the coding sequence and is operably linked to a coding sequence. As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. However, a flanking sequence does not necessarily have to be contiguous with the coding sequence, as long as it works correctly. Thus, for example, intervening untranslated transcribed sequences can still be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered operably linked to the coding sequence. Similarly, an enhancer sequence can be located upstream or downstream of the coding sequence and affect the transcription of the sequence.

In certain embodiments, it is preferred that the flanking sequence be a transcriptional regulatory region that drives high level gene expression in the target cell. The regulatory region of transcription may comprise, for example, a promoter, enhancer, silencer, repressor element, or combinations thereof. The transcriptional regulatory region can be constitutive, tissue-specific, cell type-specific (i.e., the region is driving at higher levels of transcription in one type of tissue or cell compared to another), or regulatable (ie say, sensitive to the interaction with a molecule). The source of a transcriptional regulatory region can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, so long as the flanking sequence functions in a cell causing the transcription of a nucleic acid within that cell. A wide variety of regulatory regions of transcription can be used.

Suitable transcriptional regulatory regions include, for example, the CMV promoter (ie, the CMV immediate early promoter); promoters of eukaryotic genes (ie, estrogen-inducible chicken ovalbumin gene, interferon genes, glucocorticoid-inducible tyrosine aminotransferase gene and thymidine kinase gene); and the major promoters of the early and late adenovirus gene; the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-10); the promoter contained in the 3 'long terminal repeat (LTR) of the Rous sarcoma virus (RSV) (Yamamoto, et al., 1980, Cell 22: 787-97); the thymidine kinase promoter of herpes simplex virus (HSV-TK) (Wagner et al., 1981, Proc. Natl Acad Sci. USA 78: 1444-45); the regulatory sequences of the metallothionin gene (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors such as the beta-lactamase promoter (VIIIa-Kamaroff et al., 1978, Proc. Natl Acad Sci. USA, 75: 3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl Acad Sci. USA, 80: 21-25). The specific transcription control regions of cell type and / or tissue include, for example, the control region of the elastase I gene that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639 -46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); MacDonald, 1987, Hepalology 7: 425-515); the control region of the insulin gene that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-22); the control region of the immunoglobulin gene that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-58; Adames et al., 1985, Nature 318: 533-38; Alexander et al., 1987 , Mol, Cell, Biol., 7: 1436-44); the control region of the mouse mammary tumor virus in testicular, mammary, lymphoid and mast cell (Leder et al., 1986, Cell 45: 485-95); the control region of the albumin gene in the liver (Pinkert et al., 1987, Genes and Devel., 1: 268-76); the control region of the alpha-feto protein gene in the liver (Krumlauf et al., 1985, Mol Cell. Biol., 5: 1639-48; Hammer et al., 1987, Science 235: 53-58) ; the control region of the alpha 1 -antitrypsin gene in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-71); the control region of the beta-globin gene in myeloid cells (Mogram et al., 1985, Nature 315: 338-40, Kollias et al., 1986, Cell 46: 89-94); the control region of the myelin basic protein gene in the oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48: 703-12); the control region of the myosin light chain-2 gene in skeletal muscle (Sani, 1985, Nature 314: 283-86); the control region of the gonadotropic releasing hormone gene in the hypothalamus (Mason et al., 1986, Science 234: 1372-78) and the tyrosinase promoter in melanoma cells (Hart, I. Semin Oncol 1996 February; 23 (1): 1548, Siders, et al.Cancer Gene Ther 1998 SeptemberOctober; 5 (5): 281-91), among others. It is also possible to use inducible promoters that are activated in the presence of a certain molecule or condition, such as light, heat, radiation, tetracycline or heat shock proteins, for example (see, for example, WO 00 / 10612). Other suitable promoters are known in the art.

As described above, the enhancers may also be suitable flanking sequences. Enhancers are elements of DNA that act in cis, generally about 10-300 bp in length, which act on the promoter to increase transcription. The enhancers are typically independent of orientation and position, since they have been identified both in 5 'and 3' for controlled coding sequences. Several available enhancer sequences of mammalian genes are known (ie, globin, elastase, albumin, alpha-fetus-protein and insulin). Similarly, the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancers are useful with eukaryotic promoter sequences. While an enhancer can be spliced into the vector at a position 5 'or 3' to the nucleic acid coding sequence, it is typically located at a 5 'site of the promoter. Other suitable enhancers are known in the art and would be applicable to the present disclosure.

In certain embodiments, it may be advantageous to combine a p62 polypeptide or a nucleic acid sequence encoding a p62 polypeptide, or its derivative with one or more costimulatory component (s), such as cell surface proteins, cytokines, chemokines or signage in a composition described in this document. The co-stimulatory component can be included in the composition as a polypeptide or as a nucleic acid encoding the polypeptide, for example. Suitable costimulatory molecules include, for example, polypeptides that bind to members of the CD28 family (ie, CD28, ICOS; Hutloff, et al., Nature 1999, 397: 263-265; Peach, et al., J Exp Med 1994 , 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80, Schwartz, 1992, Chen et al, 1992, Ellis, et al., J. Immunol., 156 (8): 2700-9). and B7.2 (CD86; Ellis, et al., J. Immunol., 156 (8): 2700-9); polypeptides that bind to members of the integrin family (i.e., LFA-1 (CD11a / CD18); Sedwick, et al., J Immunol 1999, 162: 1367-1375; Wulfing, et al., Science 1998, 282: 2266- 2269; Lub, et al., Immunol Today 1995, 16: 479-483) including members of the ICAM family (ie, ICAM-1, -2 or -3); polypeptides that bind to members of the CD2 family (ie, CD2, signaling lymphocyte activation molecule (CDw150 or "SLAM"; Aversa, et al., J Immunol 1997, 158: 4036-4044)) as CD58 (LFA -3; CD2 ligand; Davis, et al., Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al., Nature 1998, 395: 462-469); polypeptides that bind to the heat stable antigen (HSA or CD24; Zhou, et al., Eur J Immunol 1997, 27: 2524-2528); polypeptides that bind to members of the TNF receptor family (TNFR) (ie, 4-1BB (CD137; Vinay, et al., Semin Immunol 1998, 10: 481-489), OX40 (CD134; Weinberg, et al. Semin Immunol 1998, 10: 471-480, Higgins, et al J Immunol 1999, 162: 486-493), and CD27 (Lens, et al., Semin Immunol 1998, 10: 491-499)) as 4-1BBL ( ligand 4-1BB; Vinay et al., Semin Immunol 1998, 10: 481-48; DeBenedette, et al., J Immunol 1997, 158: 551-559), factor 1 associated with TNFR (TRAF-1; ligand 4-1BB; Saoulli, et al J Exp Med 1998, 187: 1849-1862, Arch, et al Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-1BB and OX40 ligand, Saoulli, et al. Exp Med 1998, 187: 1849-1862; Oshima, et al., Immunol 1998, 10: 517-526, Kawamata, et al., J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB and ligand OX40); Arch, et al., Mol Cell Biol 1998, 18: 558-565; Jang, et al., Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al., J Biol Chem 1998, 273: 5808-5814), OX4OL (ligand oX40, Gramaglia, et al., J Immunol 1998, 161: 6510-6517), TRAF-5 (ligand OX40; Arch, et. al Mol Cell Biol 1998, 18: 558-565; Kawamata, et al., J Biol Chem 1998, 273: 5808-5814), and C ^d 70 (CD27, ligand; Couderc, et al., Cancer Gene Ther., 5 (3): 163-75). CD154 (ligand CD40 or "CD4OL"; Gurunathan, et al., J. Immunol., 1998, 161: 4563-4571; Sine, et al., Hum. Gene Ther., 2001, 12: 1091-1102) can also be adequate

Also suitable co-stimulant components or "adjuvants" are one or more cytokines, either as polypeptides or are encoded by nucleic acids contained in the compositions of the present invention (Parmiani, et al., Immunol Lett 2000 Sep. 15; 74 (1 ): 41-4, Berzofsky, et al., Nature Immunol 1: 209-219). Suitable cytokines include, for example, interleukin-2 (IL-2) (Rosenberg, et al., Nature Med. 4: 321-327 (1998)), IL-4, IL-7, IL-12 (reviewed by Pardoll , 1992; Harries, et al., J. Gene Med. 2000, July-August; 2 (4): 243-9; Rao, et al., J. Immunol.

156: 3357-3365 (1996)), IL-15 (Xin, et al., Vaccine, 17: 858-866, 1999), IL-16 (Cruikshank, et al., J. Leuk Biol. 67 (6): 757 -66, 2000), IL-18 (J. Cancer Res. Clin. Oncol. 2001. 127 (12): 718-726), GM-CSF (CSF (Disis, et al., Blood, 88: 202-210 ( 1996)), tumor necrosis factor alpha (TNF-a), or interferons such as IFN-a, or INF- ^and other cytokines, as is known in the art, may also be suitable.

Chemokines can also be used. For example, it has been shown that fusion proteins comprising CXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor autoantigen induce antitumor immunity (Biragyn, et al., Nature Biotech, 1999, 17: 253-258). ). The chemokines CCL3 (MIP-1a) and CCL5 (RANTES) (Boyer, et al., Vaccine, 1999, 17 (Supp.2): S53-S64) may also be useful. Other suitable chemokines are known in the art.

A "signaling molecule" is a biological chemical compound involved in the transmission of information between cells. These molecules are released from the cell sending the signal, cross the gap between the cells by diffusion and interact with specific receptors in another cell, which triggers a response in that cell activating a series of reactions controlled by enzymes that lead to changes within the cell. the cell. For example, hydrogen sulfide is produced in small quantities by some cells of the human body and has a series of biological signaling functions. Only examples include nitric oxide and carbon monoxide.

It is also known in the art that immune suppressor or negative regulatory mechanisms may be blocked, resulting in improved immune responses. For example, treatment with anti-CTLA-4 antibody (Shrikant, et al., Immunity, 1996, 14: 145-155; Sutmuller, et al., J. Exp. Med., 2001, 194: 823-832), anti-CD25 antibody (Sutmuller, supra), anti-CD4 antibody (Matsui, et al., J. Immunol., 1999, 163: 184-193), the IL13Ra2-Fc fusion protein (Terabe, et al., Nature Immunol. , 2000, 1: 515-520), and their combinations (ie, anti-CTLA-4 and anti-CD25 antibodies, Sutmuller, supra) have been shown to increase antitumor immune responses and would be suitable in the practice of the present invention.

Any of these components can be used alone or in combination with other agents. For example, it has been shown that a combination of CD80, ICAM-1 and LFA-3 ("TRICOM") can enhance immune responses against cancer (Hodge, et al., Cancer Res. 59: 5800-5807 (1999). Other effective combinations include, for example, IL-12 Gm -CSF (Ahlers, et al., J. Immunol., 158: 3947-3958 (1997); Iwasaki, et al., J. Immunol 158: 4591-4601 (1997). ), IL-12 GM-CSF TNF-a. (Ahlers, et al., Int. Immunol 13: 897-908 (2001)), CD80 IL-12 (Fruend, et al. Int. J. Cancer, 85: 508 -517 (2000), Rao, et al., Supra), and CD86 GM-CSF IL-12 (Iwasaki, supra.) Any person skilled in the art will know of additional combinations useful in the present invention. with additional reagents or methods that can be used to modulate such mechanisms These reagents and methods, as well as others known to those skilled in the art, can be used in the present invention.

Other strategies can also be used to improve the efficiency of nucleic acid-based immunization, including, for example, the use of self-replicating viral replicons (Caley, et al., 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al., 2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al., 2000. Mol. Ther., 1: 497-500. Dubensky, supra; Huang, et al., 2001. J. Virol., 75: 4947-4951), electroporation in vivo (Widera, et al., 2000, J. Immunol. 164: 4635-3640), incorporation of stimulant motifs of CpG (Gurunathan, et al., Ann. Rev. Immunol., 2000, 18: 927-974; Leitner, supra; Cho, et al., J. Immunol 168 (10): 4907-13), sequences for the orientation of the ubiquitin or endocytic processing pathways (Thomson, et al.

1998, J. Virol. 72: 2246-2252; Velders, et al. 2001, J. Immunol 166: 5366-5373), VP22 sequences of the Marek's disease virus type 1 (J. Virol. 76 (6): 2676-82, 2002), primary stimulation regimes (Gurunathan, supra; Sullivan , et al., 2000, Nature, 408: 605-609, Hanke, et al., 1998, Vaccine, 16: 439-445, Amara, et al., 2001, Science, 292: 69-74), and the use of vectors. of mucosal administration such as Salmonella (Darji, et al., 1997. Cell, 91: 765-775; Woo, et al., 2001, Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below.

Chemotherapeutic agents, radiation, anti-angiogenic molecules or other agents can also be used to treat and / or prevent cancer using p62 polypeptides or nucleic acids encoding p62 (Sebti, et al., Oncogene 2000 Dec. 27; 19 (56): 6566-73). For example, in the treatment of metastatic breast cancer, useful chemotherapeutic agents include cyclophosphamide, doxorubicin, paclitaxel, docetaxel, navelbine, capecitabine and mitomycin C, among others. Combined chemotherapeutic regimens have also been shown to be effective, including cyclophosphamide methotrexate 5-fluorouracil; cyclophosphamide doxorubicin 5-fluorouracil; or, cyclophosphamide doxorubicin, for example. Other compounds such as prednisone, a taxane, navelbine, mitomycin C or vinblastine have been used for several reasons. Most patients with breast cancer have tumors with estrogen receptor positive (ER +) and in these patients, endocrine therapy (ie, tamoxifen) is preferred over chemotherapy. For such patients, tamoxifen is preferred or, as a second-line therapy, progestins (medroxyprogesterone acetate or megestrol acetate). Aromatase inhibitors (ie, aminoglutethimide and its analogs, such as letrozole) decrease the availability of estrogen needed to maintain tumor growth and can be used as second or third line endocrine therapy in certain patients.

Other cancers may require different chemotherapeutic regimens. For example, metastatic colorectal cancer is usually treated with Camptosar (irinotecan or CPT-11), 5-fluorouracil or leucovorin, alone or in combination with each other. Inhibitors of proteinases and integrin, such as the MMP inhibitors marimastata (British Biotech), COL-3 (Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (Bristol Myers Squibb), CGS 27023A (Novartis) ) or the integrin inhibitors Vitaxin (Medimmune), or MED1522 (Merck KgaA) may also be suitable for use. As such, the immunological selection of immunogenic targets associated with colorectal cancer could be performed in combination with a treatment using those chemotherapeutic agents. Similarly, chemotherapeutic agents used to treat other types of cancer are well known in the art and can be combined with the immunogenic targets described herein.

Many anti-angiogenic agents are known in the art and would be suitable for co-administration with p62 nucleic acid or polypeptide vaccines (see, for example, Timar, et al., 2001. Pathology Oncol. Res., 7 (2): 85 -94). Such agents include, for example, physiological agents such as growth factors (ie, ANG-2, NK1, 2, 4 (HGF), transforming growth factor beta (TGF-p)), cytokines (ie, interferons such as IFN-a, -p, -y, platelet factor 4 (PF-4), PR-39), proteases (ie, cleaved AT-III, collagen fragment XVIII (endostatin)), plasmin fragment HmwKallikrein-d5 (angiostatin), prothrombin-F1-2, TSP-1), protease inhibitors (ie, tissue inhibitors of metalloproteases such as TIMP-1, -2 or -3; maspin; plasminogen inhibitor activators such as PAI-1; of pigmentary epithelium (PED ^f )), Tumstatin (available through ILEX, Inc.), antibody products (ie, collagen binding antibodies HUIV26, HUI77, XL313; anti-VEGF; anti-integrin (ie , Vitaxin, (Lxsys))) and glycosidases (ie, heparinase-I, -III). Molecules that are antagonists of antigens associated with angiogenesis (including proteins and polypeptides) are also suitable and may include, but are not limited to, molecules directed against VEGF, VEGF receptor, EGFR, bFGF, PDGF-B, PD-ECGF, TGF including TGF-a, endoglin, Id proteins, various proteases, nitric oxide synthase, aminopeptidase, thrombospondins, k-ras, Wnt, cyclin-dependent kinases, microtubules , heat shock proteins, heparin binding factors, synthases, collagen receptors, integrins, and NG2 surface proteoglycan. Modified "chemical" or physiological agents that are known or believed to have anti-angiogenic potential include, for example, vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatin A, rapamycin (Guba, et al., 2002, Nature Med. , 8: 128-135), CEP-7055 (available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron, Inc.), CGS. 27023A (Novartis), tetracycline derivatives (ie, COL-3 (Collagenix, Inc.)), Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate (MTX ), irsofladin, radicicol, cyclosporine, captopril, celecoxib, sulfated polysaccharide D45152, cationic protein (protamine), cationic peptide-VEGF, suramin (naphthyl urea polysulfonated), compounds that interfere with the function or production of VEGF (ie, SU5416 or SU686)), PTK787 / ZK22584 (Novartis), Distamycin A, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives, genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid, carboxyamidotriazole, TNP-470, octreotide, 2- methoxyestradiol, aminosterols (ie, squalamine), glutathione analogues (ie, N-acetyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptor blocking agents (Nature, 414: 933-938, 2001) , Rhangiostatin, Rh-Endostatin (WO 01/93897), cyclic RGD peptide or, acutin-disintegrin, benzodiazepenes, humanized anti-avb3 antibody, Rh-PAI-2, amiloride, p-amidobenzamidine, anti-uPA antibody, anti-uPAR antibody, L-phenylalanine-N-methylamides (ie, Batimistat, Marimastat ), AG3340 and minocycline. Many other suitable agents are known in the art.

It can also be used in combination with "non-traditional" methods to treat cancer. For example, it has been shown that the administration of certain anaerobic bacteria can help slow the growth of the tumor. In one study, Clostridium novyi was modified to remove a toxin gene in a phage episome and was administered to mice with colorectal tumors (Dang, et al P.N.A.S. USA, 98 (26): 15155-15160, 2001). In combination with chemotherapy, it was shown that the treatment caused tumor necrosis in animals. The reagents and methodologies described in this application can be combined with said treatment methodologies.

The nucleic acids encoding p62 polypeptides can be administered to patients by any of the various techniques available. Various viral vectors that have been used successfully to introduce nucleic acids into hosts include retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses and smallpox viruses, among others. It is understood in the art that many of these viral vectors are available in the art. The vectors of the present invention can be constructed using standard recombinant techniques widely available to those skilled in the art. Such techniques can be found in common molecular biology references, such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D Goeddel, 1991. Academic Press, San Diego, Calif.), And PCR Protocols: A Guide to Methods and Applications (Innis, et al., 1990. Academic Press, San Diego, Calif.).

Suitable retroviral vectors include lentivirus derivatives as well as derivatives of murine or avian retroviruses. Examples of suitable retroviral vectors include, for example, Moloney murine leukemia virus (MoMuLV), Harvey's murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and sarcoma virus. de Rous (RSV). Several retroviral vectors can incorporate multiple exogenous nucleic acid sequences. As the recombinant retroviruses are defective, they require assistance to produce infectious vector particles. This assistance may be provided, for example, by helper cell lines that encode retrovirus structural genes. Suitable helper cell lines include. PSI.2, PA317 and PA12, among others. The vector virions produced using said cell lines can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large amounts of chimeric retroviral virions. Retroviral vectors can be administered by traditional methods (i.e., injection) or by implantation of a "producer cell line" near the target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5 (3): 343-79; Culver, K., et al., Cold Spring Harb. Symp; Quant. Biol., 59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). The producer cell line is designed to produce a viral vector and releases viral particles near the target cell. A portion of the viral particles released are contacted with the target cells and infect those cells, thus administering a nucleic acid of the present invention to the target cell. Upon infection of the target cell, expression of the vector nucleic acid occurs.

Adenoviral vectors have proved to be especially useful for gene transfer to eukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004): 431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42 51), the study of the expression of eukaryotic genes (Levrero, M., et al., 1991, Gene, 101 (2): 195-202), the development of vaccines (Graham, F. and Prevec, L., 1992, Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet, L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. ): 151-2; Rich, D., et al., 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routes to administer recombinant adenoviruses to different tissues in vivo have included intratracheal instillation (Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55), injection into muscle (Quantin, B ., et al., 1992, Proc. Natl Acad Sci. USA, 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard, R., 1993, Proc. Natl Acad Sci. USA , 90 (7): 2812-6) and stereotactic inoculation to the brain (Le Gal La Salle, G., et al., 1993, Science, 259 (5097): 988-90), among others.

The adeno-associated virus (AAV) demonstrates high-level infectivity, a broad range of hosts and specificity in the integration into the host cell genome (Hermonat, P., et al., 1984, Proc. Natl Acad Sci. USA, 81 (20): 6466-70). And the herpes simplex virus type 1 (HSV-1) is another attractive vectorial system, especially for its use in the nervous system due to its neurotropic property (Geller, A., et al., 1991, Trends Neurosci., 14 ( 10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4 (1): 87-99; Glorioso, et al., 1995, Annu., Rev. Microbiol., 49: 675 710).

The smallpox virus is another useful expression vector (Smith, et al., 1983, Gene, 25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al, 1992, Curr Top, Microbiol, Immunol., 158: 25-38, Moss, et al., 1991. Science, 252: 1662-1667). The smallpox viruses that are useful include vaccinia, NYVAC, fowlpox, avian diphtheria, canarypox, ALVAC and ALVAC (2), among others.

NYVAC (vP866) was derived from the Copenhagen vaccine strain of the vaccinia virus by eliminating six nonessential regions of the genome that encoded known or potential virulence factors (see, for example, U.S. Patent Nos. 5,364,773 and 5,494,807). The elimination loci were also designed as receptor loci for the insertion of foreign genes. The eliminated regions are: the thymidine kinase gene (TK; J2R); the hemorrhagic region (u; B13R B14R); the inclusion body region type A (ATI; A26L); the hemagglutinin gene (HA, A56R); the gene region of the host range (C7L-K1L); and the ribonucleotide reductase, large subunit, (I4L). NYVAC is a genetically engineered vaccinia virus strain that was generated by specifically removing eighteen open reading frames that encoded the gene products associated with the virulence and range of the host. It has been shown that NYVAC is useful for the expression of TA (see, for example, U.S. Patent No. 6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under the terms of the Budapest Treaty, entry numbers VR-2559, VR-2558, VR-2557, VR-2556, ATCC -97913, ATCC-97912 and ATCC-97914, respectively.

ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are also suitable for use in the practice of the present invention (see, for example, U.S. Patent No. 5,756,103). ALVAC (2) is identical to ALVAC (1), except that the ALVAC genome (2) comprises the vaccinia E3L and K3L genes under the control of the vaccine promoters (US Pat. No. 6,130,066; Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). It has been shown that both ALVAC (1) and ALVAC (2) are useful for expressing foreign DNA sequences, such as TA (Tartaglia et al., 1993 a, b; U.S. Patent No. 5,833,975). ALVAC was deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA U ^u ., Entry number ATCC VR-2547.

Another vector of the useful smallpox virus is TROVAC. TROVAC refers to an attenuated fowlpox that was isolated cloned in plaque derived from the FP-1 vaccine strain of fowlpox that is authorized for the vaccination of day-old chicks. TROVAC was also deposited under the terms of the Budapest Treaty with the ATCC, access number 2553.

"Non-viral" plasmid vectors may also be suitable. Suitable plasmid vectors are compatible with bacterial, insect and / or mammalian host cells. Such vectors include, for example, PCR-II, pCR3 and pcDNA3.1 (Invitrogen, San Diego, California), pBSIl (Stratagene, La Jolla, California), pET15 (Novagen, Madison, Wisconsin), pGEX (Pharmacia Biotech, Piscataway , NJ), pEGFP-N2 (Clontech, Palo Alto, California), pETL (BlueBaclI, Invitrogen), pDSR-alpha (PCT Publication No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY), as well as as plasmid derivatives Bluescript® (a phagemid based on high copy number COLE1, Stratagene Cloning Systems, La Jolla, Calif), plasmids for PCR cloning designed to clone Taq-amplified PCR products (eg, the TOPO ™ ^t A Cloning® kit, plasmid derivatives of PCR2.1®, Invitrogen, Carlsbad, California). Bacterial vectors can also be used. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guerin (BCG) and Streptococcus (see, for example, WO 88/6626, WO 90/0594, WO 91/13157, WO 92 / 1796; and WO 92/21376). Many other vectors and expression systems of non-viral plasmids are known in the art and could be used.

Suitable nucleic acid delivery techniques include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct DNA injection, CaPO4 precipitation, gene gun techniques, electroporation and colloidal dispersion systems, among others. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and administered to the cells in a biologically active form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). The composition of the liposome is generally a combination of phospholipids, particularly phospholipids with high phase transition temperatures, generally in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of the liposomes depend on the pH, the ionic strength and the presence of divalent cations. Examples of lipids useful in the production of liposomes include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Particularly useful are the diacylphosphatidylglycerols, where the lipid moiety contains from 14 to 18 carbon atoms, particularly from 16 to 18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

An immunogenic target can also be administered in combination with one or more adjuvants to stimulate the immune response. Exemplary adjuvants are shown in Table 3 below:

Table 3. Types of immunological adjuvants

In some embodiments, p62 polypeptides or nucleic acids encoding p62 according to the present invention can be used to treat, alleviate, ameliorate, placate, delay onset (prophylaxis), inhibit progression, reduce severity and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, p62 polypeptides or nucleic acids encoding p62 can be used to treat solid tumors, for example, cancer and / or cancer cells. The term "cancer" includes premalignant and malignant cancers. Cancers include, among others, prostate cancer, gastric cancer, colorectal cancer, skin cancer, for example, melanomas or basal cell carcinomas, lung cancer, breast cancer, ovarian cancer, uterine cancer, head cancers and neck, bronchial cancer, pancreatic cancer, cancer urinary bladder, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, bowel cancer thin or appendix, salivary gland cancer, thyroid cancer, cancer of the adrenal gland, osteosarcoma, chondrosarcoma, sarcoma, cancer of blood tissues and the like. The "cancer cells" may be in the form of a tumor, may exist alone within a subject (e.g., leukemia cells or ascites), or may be cell lines derived from a cancer.

Cancer can be associated with a variety of physical symptoms. Cancer symptoms usually depend on the type and location of the tumor. For example, lung cancer can cause coughing, shortness of breath, and chest pain, while colon cancer often causes diarrhea, constipation, and blood in the stool. However, to give just a few examples, the following symptoms are usually associated with many types of cancer: fever, chills, night sweats, cough, dyspnea, weight loss, loss of appetite, anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly, hemoptysis, fatigue, malaise, cognitive dysfunction, depression, hormonal disorders, neutropenia, pain, unhealed ulcers, enlarged lymph nodes, peripheral neuropathy and sexual dysfunction.

In one aspect of the invention, a method for the treatment of cancer (e.g., prostate or breast cancer) is provided. In some embodiments, the treatment of cancer comprises administering a therapeutically effective amount of p62 or p62 polypeptides encoding nucleic acids to a subject in need thereof, in such amounts and for as long as necessary to achieve the desired result. In certain embodiments, a "therapeutically effective amount" of a targeted particle is that amount effective to treat, alleviate, ameliorate, alleviate, delay the onset, inhibit progression, reduce the severity and / or reduce the incidence of one or more symptoms or characteristics of cancer.

In one aspect of the invention, there is provided a method for administering p62 or p62 polypeptides encoding nucleic acids to a subject suffering from cancer (e.g., breast cancer) or a relapse. In some embodiments, p62 polypeptides or nucleic acids encoding p62 are administered to a subject in such amounts and for as long as necessary to achieve the desired result (ie, cancer treatment). In certain embodiments, a "therapeutically effective amount" of p62 polypeptides and nucleic acids encoding p62 is that amount effective to treat, alleviate, ameliorate, alleviate, delay the onset, inhibit progression, reduce severity, and / or reduce the incidence of one or more symptoms or characteristics of cancer. In some embodiments, the p62 polypeptides or nucleic acids encoding p62 of the invention are administered to a subject previously treated for cancer. In some embodiments, p62 polypeptides or nucleic acids encoding p62 of the invention are administered to a subject with a family history of cancer. In some embodiments, p62 polypeptides or nucleic acids encoding p62 of the invention are administered to a subject with a certain predisposition for cancer. For example, a subject who is BRCA positive is genetically predisposed to certain forms of breast cancer.

The therapeutic protocols include administering a therapeutically effective amount of p62 polypeptides or nucleic acids encoding p62 to a healthy individual (i.e., a subject who does not exhibit any symptoms of cancer and / or who has not been diagnosed with cancer). For example, healthy individuals can be "immunized" with p62 polypeptides or nucleic acids encoding p62 before the development of cancer and / or the onset of cancer symptoms; individuals at risk (eg, patients with a family history of cancer, patients with one or more genetic mutations associated with the development of cancer, patients with a genetic polymorphism associated with the development of cancer, patients infected with a virus associated with the development of the cancer, patients with habits and / or lifestyles associated with the development of cancer, etc.) can be treated in a substantially contemporaneous manner (eg, within 48 hours, within 24 hours or within 12 hours). hours of the onset of cancer symptoms). Of course, people who are known to have cancer can receive innovative treatment at any time.

In other embodiments, p62 polypeptides or nucleic acids encoding p62 of the present invention can be used to inhibit the growth of cancer cells, e.g., breast cancer cells. As used herein, the expression "inhibits the growth of cancer cells" or "which inhibits the growth of cancer cells" refers to any deceleration of the rate of proliferation and / or migration of cancer cells, arrest of proliferation and / or migration of cancer cells, or death of cancer cells, so that the growth rate of the cancer cells is reduced compared to the observed or predicted rate of growth of an untreated control cancer cell. The expression "inhibits growth" may also refer to a reduction in the size or disappearance of a cancer cell or tumor, as well as to a reduction in its metastatic potential. Preferably, such inhibition at the cellular level can reduce the size, stop the growth, reduce the aggressiveness, or prevent or inhibit the metastasis of a cancer in a patient. Those skilled in the art can easily determine, by any of a variety of suitable indications, whether the growth of cancer cells is inhibited.

The inhibition of the growth of cancer cells can be evidenced, for example, by the arrest of cancer cells in a particular phase of the cell cycle, for example, the arrest in the G2 / M phase of the cell cycle. The inhibition of the growth of cancer cells can also be evidenced by direct or indirect measurement of the cancer cells or the size of the tumor. In human patients with cancer, such measurements They are usually performed using well-known imaging methods, such as MRI, computerized axial tomography and X-rays. The growth of cancer cells can also be determined indirectly, for example, by determining levels of circulating carcinoembryonic antigen, specific antigen. of the prostate or other cancer-specific antigens that correlate with the growth of cancer cells. Inhibition of cancer growth is also generally correlated with prolonged survival and / or greater health and well-being of the subject.

The compounds and compositions described herein may be administered as a pharmaceutical product or a medicament formulated with a pharmaceutically acceptable carrier. Accordingly, the compounds and compositions can be used in the manufacture of a medicament or a pharmaceutical composition. The pharmaceutical compositions of the invention can be formulated as lyophilized solutions or powders for parenteral administration. The powders can be reconstituted by the addition of a suitable diluent or other pharmaceutically acceptable carrier before use. The liquid formulations can be buffered, isotonic, aqueous solutions. The powders can also be sprayed in dry form. Examples of suitable diluents are normal isotonic saline, 5% standard dextrose in water or buffered ammonium or sodium acetate solution. Said formulations are especially suitable for parenteral administration, but may also be used for oral administration or be contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride, sodium citrate, and the like.

Alternatively, the compounds and compositions may be encapsulated, compressed or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers can be added to improve or stabilize the composition, or to facilitate the preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid vehicles include syrup, peanut oil, olive oil, saline and water. The vehicle can also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg and about 1 g per dosage unit. The pharmaceutical preparations are made following conventional pharmacy techniques which include grinding, mixing, granulating and compressing, when necessary, for tablet forms; or grinding, mixing and filling for forms in hard gelatin capsules. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. For rectal administration, the compounds of the invention can be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.

The compounds and compositions can be formulated to include other drugs or biological agents of medical utility. The compounds and compositions may also be administered in conjunction with the administration of other drugs or biological agents useful for the disease or condition to which the compounds and compositions of the invention are directed.

As used herein, the phrase "an effective amount" refers to a dose sufficient to provide sufficiently high concentrations to impart a beneficial effect on the receptor thereof. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound or composition, the route of administration, the rate of elimination of the compound or composition, duration of treatment, drugs used in combination or coincident with the compound or composition, age, body weight, sex, diet and general health of the subject, and similar factors well known in the art. arts and medical sciences. Those skilled in the art are aware of several general considerations that are taken into account in determining the "therapeutically effective amount" and are described, for example, in Gilman et al., Eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed. ., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pa., 1990. Dosage levels are generally in the range of about 0.001 to 100 mg / kg / day; with levels in the range of about 0.05 to 10 mg / kg / day are generally applicable. A compound or composition can be administered parenterally, such as intravascularly, intravenously, intraarterially, intramuscularly, intraocularly, intradermally, subcutaneously or the like. The administration can also be oral, nasal, rectal, transdermal, intravaginal or inhalation through an aerosol. A compound or composition can be administered to the organ having the tumor (or the potential target of the tumor) or to the tumor itself. The compound or composition can be administered as a bolus or infused slowly, or administered as an intradermal, subcutaneous, intramuscular or intraperitoneal injection.

A therapeutically effective dose can be estimated initially from cell culture assays by determining a level of p62 expression upon introduction of a nucleic acid encoding a p62 polypeptide. Then a dose can be formulated in animal models to achieve an adequate immune response and / or protection against tumor growth. This information can be used to determine more precisely the initial doses useful in humans. The doctor himself can choose the exact formulation, the route of administration and the dose depending on the patient's condition.

Examples

Example 1

Cell lines and vector construction

A breast tumor cell line 233-VSGA1, which overexpresses the activated rat HER / 2 neu oncogene, was derived from a mouse mammary carcinoma arising in transgenic FVB / NT NT mice. This cell line was maintained as described (Nanni P, Pupa S M, Nicoletti G et al., Int J Cancer 2000; 87: 186). HeLa cells (ATCC # CCL-2.2 ™) were propagated in complete growth medium ATCC (ATCC MD-6108).

The extracellular domain of rat HER2 / neu was amplified by PCR and cloned into the vector pcDNA3.1 (Invitrogen) as described (FM Venanzi, A Barucca, K Havas, M Capitani, M Provinciali S Scotti, A Concetti. 2010 (22); 3841-7).

As a source of cDNA encoding p62, the total RNA was extracted from HeLa cells. The full-length cDNA encoding the longest isoform of p62 (Transcript Variant 1, GenBank reference number NP-003891) was amplified by PCR (HotStar HiFidelity Polymerase Kit Qiagen) using the following primers: FW: 5-CCCGCTAGCATGGCGTCGCTCACCGTG- 3 and REV: 5'-CCCAAGCTTTCACAACGGCGGGGGATGCTTTG-3 '. The PCR products were purified and fragments digested with Nhe I-Hind III were cloned into pcDNA3.1. The inserted p62 DNA sequences were confirmed by sequencing (MGWBiotech / M-medical, Martinsried, Germany). It was observed that the encoded polypeptide differed from the natural amino acid sequence by two substitution mutations: C145R and Q418R.

Example 2. Preventive antitumor effect of p62 immunization in a breast cancer model in mouse FVB / N mice were divided into three groups (15 mice per group) and immunized with:

1. pcDNA.3.1 (empty plasmid vector, negative control);

2. pcDNA.3.1 with pHER2 (positive control); or,

3. pcDNA.3.1 with p62 (experiment).

FVB / N females were anaesthetized and, after exposure of the femoral quadriceps, 100 μg of DNA (1 mg / mL) was injected in saline with an insulin syringe. The mice were immunized twice (at 4 and 2 weeks before exposure to the tumor). Mice were stimulated intradermally with 3 x 10 5 tumor cells 233-VSGA1 / 100 pl of PBS buffer in the limb. In all cases, the tumors were measured by determining two perpendicular diameters with a calibrator three times a week. The mice were sacrificed when the tumor ulcerated or reached 1 cm in any diameter.

100% of the mice vaccinated with an empty plasmid vector (negative control) developed tumors on day 13 after exposure. Vaccination with plasmid coding for HER2 gave a protection of 40% (Figure 4). At the same time, the plasmid encoding p62 showed 100% protection on day 13, which was gradually reduced to 70%. Accordingly, the protective effect of the plasmid encoding p62 was demonstrated in a mouse model of transplantable breast cancer. Therefore, the preventive effect of the p62 vaccine was demonstrated for breast cancer in a mouse model. The inventors hypothesize that 100% protection could be maintained if the vaccines were continued.

Animals that were immunized with a plasmid vector encoding HER2 or p62 and did not develop tumors after the first cancer cell test were administered the same amount of cancer cells on day 50. All animals vaccinated with plasmid encoding HER2 they developed tumors, while no tumors appeared in animals vaccinated with p62. Consequently, immunization with p62 maintained immunological memory, whereas HER2 did not.

Example 3. The p62 vaccine stimulates innate immunity

Tumors in the mice that received the p62 vaccine contained large areas of necrosis (Figure 5). The immune cells associated with inflammation are abundantly present within the necrotic areas. The HER2 vaccine elicits antigen-specific adaptive immune response and massive migration of lymphocytes (CD3 + cells, tumor infiltrating lymphocytes) into the tumor (Figure 6). At the same time, vaccination with p62 plasmid did not significantly increase the level of tumor infiltrating lymphocytes. In contrast, the injection of plasmid p62, but not the HER2 vaccine, increased the level of CD11B + cells in the tumor (Figure 6). Consequently, the p62 vaccine acts through the stimulation of innate immunity, unlike the HER2 vaccine.

Example 4. Demonstration of the antitumor activity of the p62 DNA vaccine in the rat T5 breast cancer model

T5 transplantable rat breast cancer was derived from a spontaneous adenocarcinoma of the mammary gland of a Wistar rat in R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National Academy of Sciences of Ukraine. Two-month-old female Wistar rats (weight: 130-150 g, 10 animals per group) were challenged with T5 rat mammary gland carcinoma by subcutaneous injection of 2.5 x 10 6 tumor cells / rat in 0.4 ml of PBS. Beginning the next day after the tumor transplant, the rats were vaccinated three times once a week (Figure 7). Each injection contained 78 | jg of pcDNA.3.1 (empty plasmid vector, negative control) or pcDNA.3.1 with p62 (experiment).

Tumor growth was inhibited by immunization with p62 (Figure 8) with a 70% inhibition of tumor growth in rats vaccinated with p62 compared to control rats injected with vector (p <0.004) (Figure 9).

The survival of the rats implanted in the tumor (8 immunized with vector and 8 immunized with p62) was monitored for 75 days. 50% of the animals in the control group died while there were no animal deaths in the group vaccinated with p62 (FIG 10).

Histological analysis of the tumors revealed necrotic areas (figure 11). Intratumoral necrosis in rats receiving the p62 DNA vaccine was similar to that observed in the tumors of mice that received the p62 DNA vaccine (Figure 12).

Accordingly, the antitumor effect of the p62 DNA vaccine was demonstrated in the second animal model (rat), indicating that the p62 DNA vaccine can be used to treat breast cancer.

Example 5. Antimetastatic potency of p62 DNA vaccine

Lewis lung carcinoma is a model officially accepted by the Pharmacological Committee of the Russian Federation, "Russian FDA", to test drugs for anti-metastatic effects. 100 μg of plasmid was injected intramuscularly in each mouse 4 weeks and 2 weeks before exposure with tumor transplantation, as well as 1, 8 and 15 days after exposure. Fifteen animals were used per group. Fig. 13 shows that vaccination with p62 reduced the number of metastases in the lungs by 50% compared to the control.

Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The embodiments described illustratively herein may be suitably practiced in the absence of any element or elements, limitation or limitations, not specifically described herein. Thus, for example, the terms "comprising", "including", "containing", etc. they will be read extensively and without limitation. In addition, the terms and expressions used in this document have been used as terms of description and not limitation, and there is no intention in the use of such terms and expressions to exclude any equivalent of the characteristics shown and described or their parts, although it is recognized that various modifications are possible within the scope of the claimed invention.

Therefore, it should be understood that, although the invention has been specifically described by preferred embodiments and optional features, the modification, improvement and variation of the inventions made herein described herein may be used by the experts in the art. technique. The materials, methods and examples provided herein are representative of the preferred embodiments, are exemplary and are not intended to be limitations on the scope of the invention.

The invention has been described broadly and generically in this document. Each of the most abbreviated parts and subgeneric groups that are part of the generic description will also form part of the invention. This includes the generic description of the invention with a negative condition or limitation that eliminates any subject of the genre, regardless of whether the excised material is specifically mentioned in the present document.

In addition, when the features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group.

The above description is only intended to convey a way of understanding the present invention to those skilled in the art, and is not intended to be limiting. It will be appreciated that various modifications of the described embodiments are possible, without departing from the scope of the invention. Therefore, the scope of the present invention should be interpreted only by reference to the appended claims.

Claims (15)

1. An agent comprising: to. at least 30 consecutive amino acids of a p62 / SQSTM1 polypeptide; b. a nucleic acid encoding p62 / SQSTM1, wherein said nucleic acid encoding p62 / SQSTM1 encodes at least 30 consecutive amino acids of a p62 / SQSTM1 polypeptide; c. a p62 / SQSTM1 polypeptide at least 90% identical to SEQ ID NO. two; d. a p62 / SQSTM1 polypeptide with at least one or more domain deletions; and. a p62 / SQSTM1 nucleic acid encoding a polypeptide at least 90% identical to SEQ ID NO: 2; or f. a p62 / SQSTM1 nucleic acid encoding a polypeptide with at least one or more domain deletions; for use in the treatment, relief, improvement, palliation, delay in onset, inhibition of progression, reduction of severity and / or reduction of the incidence of one or more symptoms of a cancer in a subject. 2. The agent for the use of claim 1 (d) or 1 (f) wherein said one or more domain deletions are selected from the group consisting of PB1, ZZ, NLS2, t B, NLS1, NeS, LIR , KIR and UBA. 3. The agent for the use of claim 1 or claim 2, wherein said p62 / SQSTM1 polypeptide or p62 encoding nucleic acid is comprised in a fusion polypeptide or nucleic acid encoding a fusion polypeptide. 4. The agent for the use of any one of claims 1-3, wherein said agent is for administration with an adjuvant and / or one or more costimulatory components and / or one or more molecules that block suppressive immune mechanisms. or negative regulators; wherein said one or more costimulatory components are selected from the group consisting of cell surface proteins, cytokines, chemokines and signaling molecules; and wherein said one or more molecules that block suppressive or negative regulatory immune mechanisms are selected from the group consisting of: anti-CTLA-4 antibody, anti-CD25 antibody, anti-CD4 antibody and the IL13Ra2-Fc fusion protein. The agent for the use of claim 4, wherein said adjuvant is selected from the group consisting of: gel-like, microbial, particulate, oil-emulsion, surfactant-based and synthetic adjuvants. 6. The agent for the use of any one of claims 1-5, wherein said agent is for administration with one or more anti-cancer therapies. The agent for the use of claim 6, wherein said one or more cancer therapies is selected from the group consisting of: a chemotherapeutic molecule, radiation and an anti-angiogenic molecule. The agent for the use of claim 7, wherein said chemotherapeutic molecule is selected from the group consisting of: cyclophosphamide, doxorubicin, paclitaxel, docetaxel, vinblastine, methotrexate, navelbine, capecitabine, mitomycin C and 5-fluorouracil. The agent for the use of any one of claims 1-8, wherein said cancer is a solid tumor cancer, optionally selected from the group consisting of: breast cancer, lung cancer, prostate cancer, cancer Gastric, colorectal cancer, skin cancer, head and neck cancer, bronchial cancer, pancreatic cancer, urinary bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cancer the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, cancer of the biliary tract, cancer of the small intestine or appendix, ovarian cancer, cancer of the uterus, cancer of the salivary gland, cancer of the thyroid gland, cancer of the adrenal gland, osteosarcoma, chondrosarcoma and sarcoma, or cancer of hematological tissues. 10. The agent for the use of claims 1-9, wherein said subject is selected from the group consisting of: a subject diagnosed with cancer, a subject previously treated for cancer, a subject with a family history of cancer and a subject predisposed to cancer. The agent for the use of any of claims 1-10, wherein said agent comprises a nucleic acid encoding p62 / SQSTM1, wherein said encoded p62 protein is at least 90% identical to SEQ ID NO. . 2, and wherein said nucleic acid encoding p62 further comprises a plasmid or a viral vector. The agent for the use of claim 11, further comprising: one or more self-replicating viral replicons, one or more optimized codons, one or more CpG-stimulating motifs, one or more VP22 sequences of the Marek's disease virus type 1, one or more mucosal delivery vectors, or wherein said agent is for use in in vivo electroporation or a priming stimulation regimen. 13. A p62 nucleic acid or polypeptide vaccine comprising a p62 / SQSTM1 polypeptide or nucleic acid encoding p62 / SQSTM1 or its fragment, as defined in any one of claims 1 (a) - (f), 2 or 3 . 14. A p62 nucleic acid or polypeptide vaccine according to claim 13, further comprising an adjuvant and / or one or more costimulatory components and / or one or more molecules that block the negative regulatory or suppressor immune mechanisms as defined in the claim. 4 or claim 5. 15. A p62 nucleic acid or polypeptide vaccine according to claim 13 or claim 14, comprising a nucleic acid encoding p62 / SQSTM1, wherein said encoded p62 protein is at least 90% identical to SEQ ID No. . 2, and wherein said nucleic acid encoding p62 further comprises a plasmid or a viral vector; and / or further comprising one or more self-replicating viral replicons, one or more optimized codons, one or more CpG-stimulating motifs, one or more VP22 sequences of Marek's disease virus type 1, one or more administration vectors of the mucosa, or wherein said agent is for use in in vivo electroporation or a priming stimulation regimen.